Pipetting: A Practical Guide

Transcription

1 Pipetting: A Practical Guide Author(s): Karen Guzman Source: The American Biology Teacher, 63(2): Published By: National Association of Biology Teachers DOI: URL: %5D2.0.CO%3B2 BioOne ( is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

2 How-To-Do-It Pipetting: A Practical Guide Karen Guzman In many fields of science, including cell biology, microbiology, biotechnology, biochemistry and quantitative chemistry, pipetting is an essential skill that must be mastered. Despite this, I was surprised to find few resources, especially in the life sciences, available to the student and teacher that explain the theory and practice of pipetting in a clear and accurate manner. This motivated me to create a guide for the identification and use of pipets to accomplish three purposes. The first purpose is to clarify the identification of pipets. Many sources provide information that is inaccurate or confusing because of insufficient explanation. For example, etched rings are sometimes described as a means of identifying a blowout pipet. But do colored rings on a pipet indicate it is a blowout pipet? (The answer is no.) The second purpose is to familiarize students with the basic terminology that applies to pipets. The third purpose is to describe how to use various types of pipets. Even a single pipet is sometimes used in more than one manner. For example, push button pipets can be used in the standard or the reverse mode. This guide should help both teachers and students understand that these are both acceptable, but optional, methods of using this type of pipet. Although we can provide students with a series of steps to follow when using each type of pipet, understanding the terms and why pipets are used in certain ways will simplify the use of a variety of pipets for different purposes. The guide is divided into several sections with tables provided for easier reference. The guide is written in this manner so it can be used directly or can be easily adapted for use in the classroom. Although the information provided is not new, the guide brings Karen Guzman, Ph.D., is an Assistant Professor in the Department of Biology and Health Sciences at Meredith College, Raleigh, NC 27607; guzmank@meredith.edu. together many pieces of information to answer many questions that may arise about pipets. Because of the general nature of the information, references are not indicated in the text, but several general resources are provided at the end. Introduction The pipet is an important tool in many fields of science. But what is TD? What is a serological pipet? Even though most scientists know how to use the pipets they need, the names and markings on pipets are not always clearly understood. This guide is meant to give you brief definitions of the terms you encounter when using pipets, give you some very practical tips on distinguishing different types of pipets, and explain the use of different pipets. A glossary of terms and markings is provided in Table 1. Distinguishing Different Types of Pipets Pipets are used to transfer accurately, known volumes of liquid from one container to another. Table 2 lists some pipets commonly used, their function, and the proper method of drainage. The number of different pipets available may at first seem overwhelming. Furthermore, the type of pipet is rarely labelled on the pipet itself. Fortunately, you do not need this information to be able to use most pipets. Generally, the method of use can be determined quickly by locating information that is available on the pipet. Near the end where suction is applied to fill the pipet, you can locate several key pieces of information: (1) how the pipet is calibrated, i.e. TD or TC (2) the total volume, and increments thereof, that can be delivered, and (3) if the pipet has blowout rings that indicate a blowout type pipet. Figure 1 illustrates these and other common features of pipets. TD vs. TC The abbreviation TD or TC is marked at the end of the pipet. The temperature at which the pipet has been calibrated is also often supplied. Although most pipets you encounter will be TD, in rare circumstances you may encounter one that is TC. Since a TC pipet will contain the specified volume, it is important that you rinse out the pipet after transferring your sample and add this rinse to the portion already transferred. This ensures that any sample adhering to the inner surface of the pipet is included in the amount transferred. Volume Delivered Next, determine the volume that the pipet is able to deliver and how the pipet is graduated. This information will help you determine the method that must be used to drain the pipet. The first number indicates the total volume that the pipet will hold; the second number indicates the increments that are used for the divisions. For example, 1 ml in 1/100 means that the pipet can hold a total volume of 1 ml and that it is graduated in increments of 1/100 th of a ml. If the pipet is a fixed volume pipet, such as a volumetric, only one volume will be given since the total volume cannot be delivered in increments. In general, volumetric pipets are calibrated to be accurate to two decimal places. For example, a pipet labelled as 10 ml will transfer ml. The accuracy of specific pipets is sometimes marked on the pipet. Measuring pipets, such as a Mohr or a serological pipet, are calibrated in convenient units to allow delivery of volumes up to the maximum capacity of the pipet. Push-button pipets can be variable or fixed volume pipets and are dealt with separately below. Method of Drainage The method of drainage of a measuring pipet is easily determined by comparing the maximum capacity and the 128 THE AMERICAN BIOLOGY TEACHER, VOLUME 63, NO. 2, FEBRUARY 2001

3 Table 1. Glossary of terms and markings. Term or marking Automatic pipet Blowout pipet Colored rings Etched, frosted, or silk-screened rings Kolmer pipet Mohr pipet Ostwald-Folin pipet Push-button pipet Serological pipet Syringe TC TD Volumetric pipet Definition / Description refers to a variety of pipets that can repeatedly dispense a specified amount one type is connected to a reservoir and is operated with a pump another type ejects the specified amount from a disposable tip by motorized control of a piston the last drop of liquid is dispensed for the appropriate volume to be transferred indicates the volume of the pipet; see manufacturer s information for a color key this type of marking, when located near the top of the pipet, indicates a blowout style pipet this is not a reliable means of identifying blowout pipets because it: (1) can easily be confused with other manufacturer s markings that are used to indicate pipet size; and, (2) is not used on disposable plastic pipets which have become quite popular in fields where biological contamination is an issue. used for the Kolmer Complement Fixation technique a type of serological pipet used by draining to the baseline a type of measuring pipet used to measure variable volumes a type of volumetric pipet used as a blowout type pipet in blood serum tests a button is used to depress a spring-loaded piston that forces air out of the pipet and draws it in when the button/piston is released liquid is drawn up into a disposable non-wetting plastic tip this type of pipet can be used for macro or micro volume measurements depending on its capacity; push-button pipets that deliver micro volumes are often referred to as micropipets a type of measuring pipet used to measure variable volumes can be used to measure and transfer volumes of liquid, especially for injection in gas chromatography To contain, indicates that the pipet has been calibrated to hold (to contain) the exact volume of liquid specified To deliver, indicates that the pipet has been calibrated to deliver the specified volume of liquid when properly expelled (which depends on the type of pipet) a pipet used to measure fixed volumes sometimes referred to as a Transfer pipet graduations on the pipet. Look carefully at the divisions marked on the pipet. If you must go all the way to the end of the tip in order to deliver the total volume, the pipet is a blowout (Figure 2a). If the total volume can be delivered by draining to the last graduation marking, then drain the pipet to this baseline (Figure 2b). The method of drainage used for a volumetric pipet can be a bit more complicated to determine than for a measuring pipet. Standard volumetric pipets (Figure 2c) are drained by gravity, leaving the last drop remaining in the inside of the pipet. For some specialized types of volumetric pipets, such as the Ostwald-Folin (Figure 2d), the last drop is blown out. Manufacturers will often mark the end of reusable pipets to indicate a blowout type: A single or double ring that is etched, frosted or silk-screened onto the pipet is the marking usually used. (Note: Colored rings do not indicate a blowout pipet). For push-button pipets (Figure 2e), there are two methods of pipetting: one that uses the pipet as a blowout, the other that leaves a drop remaining in the tip. In the first method, after the desired volume is set, the plunger is depressed to the first stop, the sample is drawn up by slowly returning the push button to the up position, the sample is dispensed by depressing the plunger to the first stop, and then any residual sample is expelled by pressing the plunger to the second stop. A second method, known as Reverse Mode Pipetting, is used to reduce errors due to film retention (a thin layer of sample remaining on the inside of the pipet tip). The push button is depressed to the second stop, the sample is drawn up by slowly returning the push button to the up position, and the sample is then dispensed by depressing the plunger to the first stop. The liquid remaining in the pipet tip is not blown out. PIPETTING: A PRACTICAL GUIDE 129

4 Table 2. Pipets commonly used in science. Type or Name Function Drainage Push button or Micropipets* deliver a variable or fixed volume blow out last drop Serological, standard type deliver a variable volume blow out last drop Kolmer Serological deliver a variable volume drain to baseline Mohr deliver a variable volume drain to baseline Capillary contain a fixed volume wash out Lambda (two types) a. contain a fixed volume a. wash out b. deliver a fixed volume b. blow out Ostwald-Folin deliver a fixed volume blow out last drop Volumetric, standard type deliver a fixed volume drain by gravity leaving last drop in pipet *Push button pipets can be divided into two types that differ in their mechanics: air displacement and positive displacement. Air displacement pipets are used for routine measurements while positive displacement pipets have a greater accuracy and precision and are preferred for use with samples having a high viscosity, surface tension, density, or vapor pressure. Using Different Types of Pipets Two characteristics are important to determine before using any pipet. As described above, first: determine if the pipet should be drained to the baseline, drained until the last drop remains in the tip of the pipet, or if all of the liquid must be blown out of the pipet. Second: determine the volume, and the increments thereof, that can be delivered. Now you are ready to pipet a specified volume. Figure 1. The parts of a pipet. (a) A type of measuring pipet is used to illustrate several common features of pipets including (1) the end to which a pipet aid is attached to apply suction; (2) blowout rings that when present indicate a blowout type pipet: etched, frosted, or silk-screened rings are generally the type of markings used to indicate a blowout pipet; (3) color rings that indicate the volume of the pipet; and (4) calibration information. (b) A push button pipet is used to illustrate features common to this type of pipet including the (1) push button used to draw up and eject liquids, (2) volume indicator that displays the volume to which the pipet has been set, and (3) disposable tip into which the liquid is drawn. Liquid enters and exits the pipet through the orifice of the tip, which is indicated in the figure for each of the pipets shown. 1. To rinse the interior surface of the pipet, draw a portion of the liquid to be sampled into the pipet once or twice and return the rinse to the sample. Be sure to insert the pipet only a short distance into the liquid so that the sample does not adhere to the outer surface of the pipet and cause inaccurate measurement. 2. Now draw the specified volume into the pipet using a suction bulb or other pipet aid. (For push-button pipets, the plunger fulfills this function.) Be sure no bubbles are in the liquid in the pipet. If necessary, any liquid remaining on the outside of the tip should be wiped away, taking care not to touch the orifice of the pipet tip (Figure 1). 3. Touch the tip of the pipet to the inner wall of the vessel to which you wish to transfer the sample. Drain the sample using one of the following methods: a. For pipetting of multiple volumes, drain the specified amount until the bottom of the meniscus moves from one graduation to the next appropriate 130 THE AMERICAN BIOLOGY TEACHER, VOLUME 63, NO. 2, FEBRUARY 2001

5 marking. Follow directions below for the final drop. b. For pipets that have a baseline, continue to drain until the liquid reaches the baseline. Discard the final portion of liquid remaining in the pipet. c. For volumetric and non-blowout pipets, allow the liquid to drain by gravity. Continue to hold the tip of the pipet against the wall of the vessel for about 10 seconds. Withdraw the pipet with a rotating motion to remove any droplet still adhering to the tip. The small volume remaining inside the tip can be discarded. d. For blowout pipets, drain the entire contents of the pipet, keeping the tip of the pipet against the wall of the vessel. To eject the drop adhering to the inside of the pipet, blow air through the pipet with the pipet aid as you draw the pipet away from the surface of the vessel. (This allows surface tension to help remove this last drop of liquid from the pipet.) If necessary, blow air through the pipet again to evacuate the pipet. Figure 2. Common types of pipets used in science. Several common types of pipets are illustrated in the figure. Notice several details from the figure. (a) Serological, standard type: often the graduation marks continue to the end of the pipet. (b) Mohr: drainage of this pipet is to the baseline, in this case marked by a 10. (c) Volumetric, standard type: the single graduation mark indicates the fill mark and the colored band near the suction end of the pipet indicates the volume. (d) Ostwald-Folin: the single graduation mark indicates the fill mark; the etched rings near the suction end indicates this is a blowout pipet. (e) Push button: the numeric display indicates the volume the pipet is set to deliver; a disposable plastic tip is attached at the end opposite of the push button. References Christian, G.D. (1994). Analytical Chemistry, 5 th ed. New York: John Wiley & Sons, Inc. Fisher Scientific Catalog. (1998/1999). Pittsburgh, PA: Fisher Scientific, U.S. Headquarters. Instructions for Pipetman (pamphlet). (1993). Woburn, MA: Rainin Instrument Company, Inc. Skoog, D.A., West, D.M. & Holler, F.J. (1990). Analytical Chemistry: An Introduction, 5 th ed. Philadelphia: Saunders College Publishing. PIPETTING: A PRACTICAL GUIDE 131